建構大腸菌之周質腔與胞外表達系統

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標題:	建構大腸菌之周質腔與胞外表達系統 Construction of periplasmic and extracellular expression systems in E. coli
作者:	陳韋仁
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所江建民
關鍵字:	周質腔胞外 YebF YebF extracellular periplasmic
日期:	2009
上傳時間:	2011-10-27 14:43:07 (UTC+8)
摘要:	為了研究將目標蛋白表達至大腸菌的周質腔，並進一步運送到大腸菌胞外培養液中，我們嘗試建構大腸菌的周質腔與胞外的表達系統，並測試其表達能力。我們建構了 pET21-YebF 、pET21-YebFEZZ、pET21-6HisYebF、pET26-DsbA、pET26-DsbC、pET26-Crr周質腔與胞外的表達質體。其中，pET21-YebF、pET21-YebFEZZ、pET21-6HisYebF、pET26-DsbA、pET26-DsbC，能成功的表達至周質腔並進一步送出至胞外。這些表達系統被用來檢測具有雙硫鍵的human epidermal growthfactor (hEGF)及Cardiotoxin A3 (CTX A3)的表達情形。hEGF在這些表達系統中都可被表達至胞外，以有高水溶性融合伴的情形下表達最好。而沒有任一組別在誘導後在SDS-PAGE觀察到相對應的融合CTXA3。經探討含CTX A3各組在誘導後的質體穩定性，發現含有質體的菌比例隨著誘導時間迅速減少，暗示表達的CTX A3會造成菌體死亡。 � To express the recombinant protein into periplasmic space, and furthersecret into extracellular mediumin, in E. coli, the expression vectors,pET21-YebF, pET21-YebFEZZ, pET21-6HisYeb, pET26-DsbA, pET26-DsbC,pET26-Crr were constructed and test. The results show that all fusion tag can be expressed into perimplasmic space. Two disulfide-bond containing proteins, human epidermal growth factor(hEGF) and snake cardiotoxin A3 (CTX A3), were test in these expression systems. hEGF can be expressed into perimplasmic space in E. coli in all vectors test. The suitable vectors are those containing soluble fusion tag, pET-yebF-EZZ,pET-DsbA, and pET-DsbC. None of the bands corresponding CTX A3 fusion protein were seen in SDS-PAGE. The CTX A3 containing plasmid lost severly after the induction. It implies that the expression of CTX A3 causes the death of expression host.
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显示于类别:	[生物科技系(所)] 博碩士論文

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